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Cite this:DOI: 10.1039/c6sm02208a

3D printing of self-assembling thermoresponsivenanoemulsions into hierarchicalmesostructured hydrogels

Lilian C. Hsiao,† Abu Zayed Md Badruddoza, Li-Chiun Cheng and Patrick S. Doyle*

Spinodal decomposition and phase transitions have emerged as

viable methods to generate a variety of bicontinuous materials.

Here, we show that when arrested phase separation is coupled

to the time scales involved in three-dimensional (3D) printing

processes, hydrogels with multiple length scales spanning nano-

meters to millimeters can be printed with high fidelity. We use an

oil-in-water nanoemulsion-based ink with rheological and photo-

reactive properties that satisfy the requirements of stereolitho-

graphic 3D printing. This ink is thermoresponsive and consists of

poly(dimethyl siloxane) droplets suspended in an aqueous phase

containing the surfactant sodium dodecyl sulfate and the cross-

linker poly(ethylene glycol) dimethacrylate. Control of the hydrogel

microstructure can be achieved in the printing process due to the

rapid structural recovery of the nanoemulsions after large strain-

rate yielding, as well as the shear thinning behavior that allows the

ink to conform to the build platform of the printer. Wiper operations

are used to ensure even spreading of the yield stress ink on the

optical window between successive print steps. Post-processing of

the printed samples is used to generate mesoporous hydrogels that

serve as size-selective membranes. Our work demonstrates that

nanoemulsions, which belong to a class of solution-based materials

with flexible functionalities, can be printed into prototypes with

complex shapes using a commercially available 3D printer with a few

modifications.

1. Introduction

Self-assembly is a popular method to engineer hierarchicalmicrostructure in many types of soft materials including polymerichydrogels.1 Hydrogels can be formed through chemical or physicalcrosslinking methods, and are particularly attractive because theyprovide a hydrated environment with tunable diffusion profiles

and mechanical properties.2–7 Existing strategies to preparestructured hydrogels include the use of freeze drying8,9 andsacrificial templating.10–12 These methods only provide a coarselevel of control over the resultant porous microstructure.Spinodal decomposition and phase transitions in situ have recentlyemerged as viable methods to generate a variety of bicontinuousmaterials including bijels,13 demixing polymers,14 and nematicliquid crystals.15 These multiphase systems display a rich varietyof microstructure due to the non-equilibrium thermodynamicinstability of the constituent building blocks. Temperatureresponsiveness is inherent to many of these systems andrepresents a simple and effective tuning parameter to accessmicrostructure in the micron length scale. Responsive colloidsthat form gel networks represent a particularly effective methodof self-assembling such types of structure. Their characteristicdomain size depends on the thermodynamics and kinetics ofthe highly non-equilibrium process.16,17 Large, heterogeneousvoids with strands that range from hundreds of nanometers totens of microns are commonly observed in colloidal gels.18

Here, we show that when the dynamics of gelation arecoupled to the time scales involved in three-dimensional (3D)printing processes, hydrogel materials with multiple lengthscales spanning nanometers to millimeters can be printedrapidly. 3D printing has gained popularity in recent years as anadditive manufacturing platform that offers unparalleled flexibilityin creating structures with arbitrary shapes and materialproperties.19,20 Compared to ink jetting and extrusion-basedmethods, stereolithographic (SLA) printing is a layer-by-layerprototyping technique that shows excellent compatibility withliquid polymeric materials. A traditional SLA 3D printer operatesby photopolymerization at the surface of a resin reservoir, inwhich the constructed model is progressively built by movingdownwards into the ink bath.21 Modern SLA 3D printers utilizean inverse setup and a layer-by-layer process to improve theprinting resolution of each printed layer. First, a build platformis immersed into the resin tank containing the precursor ink. Themicrostructure is then photopolymerized using a 405 nm laserand actuated micromirrors, which patterns the desired motif

Department of Chemical Engineering, Massachusetts Institute of Technology,

Cambridge, MA 02139, USA. E-mail: [email protected]

† Present address: Department of Chemical and Biomolecular Engineering,North Carolina State University, Raleigh, NC 27695, USA.

Received 28th September 2016,Accepted 23rd December 2016

DOI: 10.1039/c6sm02208a

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Soft Matter This journal is©The Royal Society of Chemistry 2017

through an optically transparent window. Finally, the platform iswithdrawn and re-immersed again to print the next layer, withautomated wiper actions between each print step to removeremnant debris on the optical window (Fig. 1). Oxygen inhibitionof the free radical polymerization is an important parameterto monitor as it controls the overall printing speed.22 Anoxygen permeable poly(dimethyl siloxane) (PDMS) layer istypically placed above the optical window to ensure a thinlubrication layer between the printed model and the resintank, which prevents the model from adhering permanentlyto the window.19,23,24

In order to leverage SLA printing for the fabrication of self-assembled hydrogels, the rheological properties of the inkshould be compatible with the movement of the build platform.High strain rate deformations are applied to the ink as theplatform is repeatedly immersed and retracted over the durationof the printing process. Many viscoelastic self-assembledmaterials can exhibit fluid instabilities25 and yield irreversiblyunder these types of large stresses.26 Thus, in order to print 3Dmesostructured hydrogels with high speed and fidelity, theink must be able to recover its original microstructure rapidlyafter yielding. In addition, the material should exhibit liquid-like response27 during platform movement such that itconforms to the base of the printed motif. Here, we demon-strate that thermoresponsive nanoemulsions, which belongto a class of multiphase materials, can be printed intohierarchical structures using a modern 3D SLA printer whenthe rheology of the complex fluid during the printing process isaccounted for.

2. Experimental sectionMaterials

We present a oil-in-water nanoemulsion-based ink with rheologicaland photoreactive properties that satisfy the requirements of SLA3D printing. Chemicals are purchased from Sigma-Aldrich andused without further purification unless noted. The nanoemulsionsconsist of a continuous phase with 33 vol% poly(ethylene glycol)dimethacrylate (PEGDMA, molecular weight Mn = 750 g mol�1) and200 mM sodium dodecyl sulfate (SDS) dissolved in deionized (DI)water, as well as an oil phase consisting of poly(dimethyl siloxane)(PDMS) droplets (viscosity, Z = 5 cP) suspended at a volume fractionof f = 0.25. The hydrophobic fluorescent dye Nile red (excitationwavelength = 550 nm, emission wavelength = 626 nm) is dissolvedin the PDMS at a concentration of 0.05 mg mL�1 to enable confocalmicroscopy imaging of the nanoemulsions.

To synthesize the nanoemulsions, a crude emulsion is firstprepared by adding the oil phase into the continuous phaseunder stirring. The crude emulsions are then passed through ahigh pressure homogenizer (Emulsiflex-C3, AVESTIN) at a pressureof 15 000 psi for 20 passes. A heat exchanger is used to chill thesample to 5 1C between each pass. After homogenization, thenanoemulsions are placed in a centrifuge at 5000 rpm to removeany large impurities and the supernatant is stored at 4 1C untilfurther use. The final diameter of the droplets is measured usingdynamic light scattering (90Plus PALS, Brookhaven Instruments)after dilution to f = 0.002 with a solvent of 33 vol% PEGDA in DIwater (Z = 5.8 cP). The diameter of the nanoemulsion dropletssynthesized using this homogenizing method is 2a = (40� 8) nm.28

Fig. 1 3D printing of self-assembled thermoresponsive nanoemulsion inks. (A and B) Schematic of commercially available stereolithographic (SLA)printer with a custom-modified Teflon window to enhance oxygen permeability. Layer-by-layer SLA printing consists of four steps in which the desiredmotif is photocrosslinked, followed by high strain-rate withdrawal and submersion steps. The process repeats at a z-step size of 100 mm.(C) Nanoemulsions (yellow) stabilized by surfactants in the resin tank are heated to induce self-assembly through interdroplet bridging of PEGDMAgelators (red are dimethacrylate groups). (D) 3D honeycomb and woodpile structured hydrogels formed by using the nanoemulsion inks. (E and F) Theinternal morphology of the 3D printed scaffold is either homogeneous at T o Tgel, or interconnected when T Z Tgel.

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In order to synthesize the SLA printer ink, we require aphotoinitiator additive that is compatible with the nanoemulsionsand that is tailored to the wavelength of the laser source. Thephotoinitiator 2-hydroxy-2-methylpropiophenone (Darocur) istypically used in photocuring applications. However, we findthat the nanoemulsion droplet size becomes unstable andincreases significantly when Darocur is incorporated (Fig. 2).An alternative photoinitiator is phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (TPO), which exhibits significant absorbance inthe l = 385 to 420 nm wavelength range and high molarextinction coefficients.29 TPO is commonly used in photocurableresins because of the overlap between its absorbance spectrumand the emission wavelength of the near-UV light sources in 3Dprinting. We find that adding 0.1 g mL�1 of TPO in a 2 vol%co-solvent of butyl acetate provides the best balance betweensolubility, nanoemulsion stability and photocurability. The dropletsize remains stable over a period of 2 h, which is comparable tothe total print time. The photoinitiator additive is added to thenanoemulsion suspension at 2 vol% to generate fresh samplesprior to each 3D printing experiment.

SLA printer modifications and setup

During the polymerization step in the chemical crosslinking ofPEGDMA, the presence of oxygen slows the reaction by scavengingreactive photoinitiator radicals.22 While this is normally undesirable,having a sufficiently thick oxygen inhibition layer below the curezone is necessary to avoid scaffold adhesion to the opticalwindow.19,23,24 We modify the resin tank of a commerciallyavailable desktop SLA printer (Form 2, FormLabs) by replacingthe PDMS layer above the optical window with a thin, opticallytransparent Teflon coating to ensure a sufficient lubricationlayer resulting from the unpolymerized ink.19 Teflon is effectivebecause it has one of the highest oxygen permeabilites in polymericmaterials (1600 barrers = 1.2 � 10�14 m2 s�1 Pa�1 30) that is doublethat of PDMS (800 barrers = 0.6 � 10�14 m2 s�1 Pa�1 31). Theprocedure to modify the resin tank is as follows. After removal ofthe PDMS layer, the surface of the window is treated with a highfrequency electrode gun (BD-10AS, Electro-Technic Products).

The plasma-treated window is evenly coated with 1H,1H,2H,2H-perfluorooctyltrimethoxysilane (Matrix Scientific) and baked inan oven (T = 80 1C) for at least 8 h. The window is then cleanedwith pure ethanol, dried, and coated with a layer of 1 wt%poly(4,5-difluoro-2,2-bis-trifluoromethyl-1,3-dioxole-co-tetra-fluoroethylene) (Teflon AF 2400) in tetradecafluorohexane(FC-72). After the Teflon coating is completely dried, theedges of the optical window are reinforced with a UV adhesive(NOA 65, Norland Optics) to prevent nanoemulsion ink leakageinto the optical chamber of the 3D printer.

To print hierarchical materials using the thermoresponsivenanoemulsions, the 3D printer is placed in an environmentalincubator (Model 3110, Thermo Forma) set to six differenttemperatures (T = 22.0 1C, 28.5 1C, 31.5 1C, 34.0 1C, 38.0 1C,39.5 1C). The setup is allowed to equilibrate for at least 2 h priorto starting the printing experiment. Approximately 250 mL ofthe freshly prepared nanoemulsion ink is loaded into the resintank and allowed to rest quiescently for 10 min to inducethermogelation. Samples used for confocal microscopy andscanning electron microscopy imaging are printed as slabs(20 mm � 80 mm � 1.5 mm) with 15 layers (thickness = 100 mmper layer). The total print time for each slab is 32 min.

Rheological characterization

Rheological measurements on the nanoemulsions are carriedout using a stress-controlled rheometer (AR-G2, TA Instruments)equipped with an aluminum 21 cone-and-plate geometry (diameter =60 mm, truncation gap = 58 mm). Small amplitude oscillatorymeasurements (strain g = 5 � 10�4, angular frequencyo = 20 rad s�1) are used in the temperature ramp (ramp rate =0.5 1C min�1) and temperature jump studies (T = 22.0 1C,30.0 1C, 35.0 1C, 40.0 1C, 45.0 1C). The linear viscoelasticmoduli, G0 and G00, are monitored over the duration of theoscillatory measurements. In order to avoid slip, ultra finesandpaper (320 grit) is adhered to the top geometry and thebottom Peltier plate for stress sweep experiments (g = 1 � 10�4

to 15, o = 20 rad s�1), repeated yielding/recovery experiments(g = 10, o = 20 rad s�1 and Dt = 20 s for the yield step; g = 5� 10�4,o = 20 rad s�1 and Dt = 80 s for the recovery step), as well assteady shear experiments (shear stress s = 0.01 to 100 Pa) thatmeasure the change in nanoemulsion viscosity as a function ofshear rate. We use pure nanoemulsions that has been passedthrough a 1.5 mm nylon filter in the rheological studies. Atemperature ramp oscillatory experiment on nanoemulsionscontaining 2 vol% of the photoinitiator mixture is conducted tocheck for consistency in the gelation temperature Tgel.

Sacrificial templating and bead infusion studies

The nanoemulsions are removed from the printed hydrogelsusing a gradual solvent transfer process. Briefly, samples arerinsed with pure ethanol, transferred into a 1 : 1 v/v isopropanol/ethanol mixture for 30 min, followed by a first soak in puretoluene for 2 h. The toluene bath is replaced with fresh solventand samples are allowed to sit for at least 12 h to ensurecomplete removal of the PDMS, which is verified by a loss influorescence. To re-hydrate the samples, a reverse solvent transfer

Fig. 2 Change in PDMS droplet size as a function of time for variousphotoinitiator additives. The additives are: 2 vol% butyl acetate (black opencircles), 2 vol% butyl acetate with 0.067 g mL�1 TPO (black open dia-monds), 2 vol% butyl acetate with 0.1 g mL�1 TPO (red filled circles), and2.9 vol% hexanediol diacrylate with 0.08 g mL�1 TPO and 0.08 g mL�1

Darocur. Error bars represent the polydispersity of the droplets computedfrom fitting the raw autocorrelation data from dynamic light scatteringwith a log-normal distribution.

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into 1 : 1 v/v isopropanol/ethanol, 1 : 1 v/v ethanol/DI water, andfinally pure DI water is used. Samples are kept in DI water for atleast 24 h to swell to their equilibrium state.

Fluorescent carboxylated polystyrene beads (5 wt%, diameters =50 nm, 200 nm, and 1.0 mm, excitation/emission wavelengths =505/515 nm) are purchased from Invitrogen and used withoutfurther purification. We place the porous hydrogel samples in200 mL of the bead suspension for 4 h. After rinsing with DIwater, the samples are ready for confocal microscopy imaging.

Microscopy imaging

We use confocal laser scanning microscopy (CLSM) to visualizethe internal structure of the printed samples containing nano-emulsions and those containing fluorescent beads after sacrificialtemplating. Imaging is performed on a Nikon A1R CLSM equippedwith a resonant scanner head, diode lasers emitting at 488 and561 nm, and a 60� oil immersion objective (NA = 1.4, workingdistance = 13 mm). Samples with dimensions of 5 mm � 5 mm �1 mm are directly cut from the printed hydrogels and placed on a#1.5 coverslip for imaging in the xy- and xz-planes. The xy-planeimages used for structural analysis have dimensions of 60.9 mm �60.9 mm with a pixel size of 119 nm. A fast Fourier transform (FFT)is applied to the raw images using the image processing softwareImageJ (NIH). The radially averaged scattered light intensity ofthe FFT images, I(q), is obtained using a radial profile plugin toImageJ.

Porous hydrogel samples (T = 22 1C, 34.0 1C) are also imagedwith a high resolution scanning electron microscope (HRSEM,Zeiss) at 5 kV accelerating voltage. Samples are cut into thin(B1 mm) sections, dried for at least 48 h at room temperature,and then adhered with carbon tape onto SEM stubs. All samplesare sputter coated with approximately 10 nm of a Au–Pd alloyprior to imaging.

Mechanical characterization of filled and porous hydrogels

Nanoemulsion inks are photopolymerized into a traditionaldogbone shape (cross sectional area, A0 = 1.00 � 10�5 m2,testing area length, L0 = 15 mm, thickness = 2 mm) for stress–strain mechanical testing. Briefly, 800 mL of nanoemulsions isadded into a dogbone-shaped mold made out of a transparentpolycarbonate sheet. A microscope coverslip is used to cover thetop of the mold. The filled mold is placed in a vacuum oven(heated to the various temperatures used in the 3D printing)purged with nitrogen gas in order to remove oxygen fromthe samples. This permits even curing during the photo-polymerization and reduces the formation of air bubbles. Afteran hour, the mold is placed under a UV lamp for 5 minutes tofully photopolymerize the hydrogels. To generate porous hydrogelsfor mechanical testing, we further treat the dogbones with tolueneand rehydrate using the procedure that is described in theexperimental section under sacrificial templating.

Both ends of the hydrogel dogbones are attached to cardboardscaffold using UV glue (NOA 65, Norland Optics). The cardboardpieces are clamped directly onto a set of 10N load cells on an Instron8840 MicroTester. The testing procedure involves the application ofan uniaxial extensional strain at a rate of 0.01 mm s�1 until rupture

occurs. Young’s modulus is obtained using the relation, E = se/e =(FL0)/(A0DL), where se is the tensile stress, e is the applied strain, F isthe applied load, L0 and A0 are the original dimensions of thehydrogel dogbone, and DL is the deformation length measured bythe Instron tester. The engineering rupture stress is obtained usingthe relation srup = Frup/A0 where Frup is the measured force at whichthe sample fractures. A minimum of six independently preparedsamples are used to generate each data point.

3. Results and discussionPrinting of self-assembled nanoemulsion hydrogels

The schematic in Fig. 1A and B shows the dynamical processused to print nanoemulsion-filled hydrogels with macroscopicfeatures (honeycomb and multi-tier woodpile hydrogels areshown in Fig. 1D). Using temperature as a tuning parameter,we are able to print hydrogels that have internal microstructurethat range from nanometer-sized droplets to mesoscopic structures.At T o Tgel, the nanoemulsions are photo-crosslinked into ahomogeneous matrix. When the temperature is raised beyond acritical gelation point Tgel, the hydrophobic end groups on thePEGDMA molecules partition into the oil phase and serve asinterdroplet bridges32 (Fig. 1C), leading to the formation of aviscoelastic colloidal gel with different microstructures (Fig. 1Eand F). Depending on application needs, these domains caneither serve as pathways for the transport of hydrophobicmolecules or be extracted to leave behind interconnected pores.

Rheological properties of the nanoemulsion inks

The viscoelasticity of the gelled nanoemulsions is an inherentmaterial property that is directly correlated with the stress-bearing microstructure and dynamics of the gel network.33,34 Assuch, the internal length scale of the printed hydrogel scaffoldcan be estimated a priori by measuring the elastic (G0) andviscous moduli (G00) of the nanoemulsions as a function oftemperature using small amplitude oscillatory rheology.

The temperature ramp experiments shown in Fig. 2 areperformed at a heating rate of 0.5 1C min�1 to generate highresolution viscoelastic data at small temperature intervals. Astemperature increases, G0 and G00 increase rapidly and reach aplateau at high temperatures. The point at which G0 = G00 setsthe value of Tgel = 29.2 1C for this particular nanoemulsionsystem, although the gel temperature can be easily adjusted byreplacing PEGDMA with other crosslinking molecules with twoor more hydrophobic end groups.32 To account for the effect ofthe heating rate, we perform temperature jump measurementsthat are representative of print conditions. Fig. 3A indicatesthat G0 and G00 follow the same trend as the temperature rampexperiments, and that Tgel is found within a similar range. Therheology data shown in Fig. 3A and 4B–D are generated usingpure nanoemulsions. Addition of the photoinitiator additiveresults in a minor reduction in Tgel to 27.6 1C (Fig. 3B), althoughthe overall rheological behavior remains similar.

In order to characterize the rheology of the nanoemulsioninks during the printing process, we estimate the compressive


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strain, g, as the relative vertical displacement of the fluidinduced by the platform movement, and the strain rate, _g,using the linear velocity of the motorized stage through the ink(Fig. 4A). The motorized platform moves at an average speed of1.27 cm s�1 in the withdrawal and re-submersion steps (steps 2and 3 in Fig. 1A). There is a constant gap of hgap = 100 mm, set bythe z-resolution of the printer, between the optical window andthe bottom of the printed model. The total height of the fluidstays relatively constant at htotal B 7.5 mm. Yielding of the gelsoccurs at strain units (g o 0.01) lower than the deformationimposed during the print process by the build platform in theprinter (g B 0.98).

Fig. 4B shows that there is a transition from the linear Glin0

at low g to the nonlinear regime beyond the yield strain gy,where G0 decreases by more than three orders of magnitude.We note here that the angular frequency (o) of the oscillatoryrheology is set to 20 rad s�1 to generate high resolution data onthe rheometer. This value of o provides the correct values G0

and G00 particularly when they become insensitive to differencesin o above the gel point of a viscoelastic suspension.35 We userepeated cycles of large amplitude oscillatory shear (LAOS) andsmall amplitude oscillatory shear (SAOS) to investigate thestructural recovery of the inks under the deformation imposedduring the printing process. In the LAOS cycle, a strain amplitudemuch larger than the yield strain of the nanoemulsion isapplied to simulate the withdrawal and submersion steps ofprinting. In the SAOS cycle, a small strain is used to probe theviscoelastic recovery of the ink during the print step. Thetimescales and parameters of the LAOS and SAOS cycle arematched to the printing step timescales as shown in Fig. 1A.Fig. 4C shows that the gelled nanoemulsions recovers to itsoriginal state rapidly and reversibly once the deformationceases. This structural recovery is key to printing hydrogelswith continuous and well-controlled internal microstructure inall three dimensions.

Within the operation range of the moving build platform( _g = 1.7 s�1), the nanoemulsions undergo shear thinning with aviscosity upper range of B100 Pa s (Fig. 4D). The yield stress, sy,and the power law exponent, n, can be obtained from fitting theHerschel–Bulkley model s = sy + k _gn to the viscosity data, wherek is a consistency parameter and the steady shear viscosity isZ = s/ _g.36 A purely Newtonian liquid has constant viscosity at allshear rates (n = 1), whereas 0 r n o 1 for a shear thinningfluid. These material properties are shown in Table 1 fornanoemulsions at different temperatures. In addition, we per-form steady state characterization of the thermoresponsivenanoemulsions at T = 22 1C and 40 1C, which encompassesthe full range of conditions tested in our study. We observesome viscosity hysteresis of the nanoemulsions at T = 22 1C,

Fig. 4 Rheology of thermoresponsive nanoemulsions. (A) Schematic ofthe motorized build platform imposing a compressive strain at a knownshear rate on the nanoemulsion ink. For our printing setup, g = 0.98 and_g = 1.7 s�1. (B) Stress sweep experiments determines the linear regime andthe yield strain at T = 22 1C (black), 30 1C (red), 35 1C (orange), 40 1C(green) and 45 1C (blue). Solid lines guide the eye. (C) Repeated largeamplitude and small amplitude oscillatory experiments show that nano-emulsions recover rapidly and reversibly. Dashed lines mark the bound-aries between yielding and recovery steps. The time scales and applieddeformations are chosen to match the print and retraction steps in the 3Dprinter. Solid symbols represent G0 and open symbols represent G00.(D) Steady shear rheology show yield stress behavior and shear thinningover the range of shear rates probed. Solid lines are Herschel–Bulkley fits.

Fig. 3 Temperature responsiveness of the nanoemulsion inks. (A) Temperatureramp experiments captured at a heating rate of 0.5 1C min�1 (purple) are overlaidwith viscoelasticity data from the linear regime in the stress sweep measure-ments (squares), fully recovered samples in the yielding/recovery measurements(circles), and discrete temperature jump experiments (down triangles).(B) Comparison of the values of Tgel for samples with (red) and withoutthe photoinitiator additives (blue). Solid symbols represent G0 and opensymbols represent G00.


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but none at T = 40 1C (Fig. 5). This gradual increase in viscosityover multiple cycles at low temperatures could be explained bythe formation of flow-induced clusters from the Brownian

relaxation of the nanoemulsions and structural relaxation ofthe PEGDMA matrix with respect to viscous forces.37 Nevertheless,the printed microstructure is unlikely to be affected by the shearimposed by the build platform given the long equilibration timeduring the print step. The yield stress of the material does presentissues if the print area is not properly refilled after the withdrawalof the build platform. Classical Saffman–Taylor instabilitiescan also lead to viscous fingering that are comparable to thelength scale of the self-assembled gel network within thenanoemulsions.38 Thus, wiper operations are necessary tospread the ink out evenly between each layer-by-layer cycle inthe printing process.

Hydrogel microstructure and mechanical properties

Fig. 6A is a panel of representative confocal laser scanningmicroscopy (CLSM) images in the xy-plane that show the rangeof microstructures of the hydrogels printed with the self-assembling nanoemulsions. Visual inspection shows that thescaffold is mostly homogeneous below the gel temperature atT = 22.0 1C. Finely percolated networks form near the gel pointat T = 28.5 1C, which develop into spinodal-like structures atT = 34.0 1C. As the temperature is further increased toT = 39.5 1C, arrested phase separation results in a decrease inthe length scale of the colloid-rich regions. We compute thescattered intensity I(q) from a fast Fourier transform (FFT)applied to the raw 2D images (Fig. 6B), where q represents thewave vector or inverse length in real space. The characteristic gel

Table 1 Rheological properties of nanoemulsion inks

T (1C) sy (Pa) n Glin0 (Pa)

22.0 0.06 0.75 0.1730.0 0.08 0.41 3.2435.0 22 0.25 899.440.0 25 0.30 222445.0 28 0.28 2210

Fig. 5 Viscosity hysteresis in thermoresponsive nanoemulsions. Steadystate viscosity plotted as a function of shear rate for four upwards (uptriangles) and downwards (down triangles) stress sweep cycles at T = 22 1C(red) and 40 1C (blue). Dashed line represents instrument sensitivity limits.

Fig. 6 Microstructure of 3D printed hydrogels using thermoresponsive nanoemulsions. (A) Representative 2D confocal laser scanning microscopy(CLSM) images of the internal microstructure of printed hydrogels, where fluorescent regions belong to poly(dimethyl siloxane) (PDMS) droplets.(B) Intensity I(q) as a function of the wave vector q, for samples printed at T = 22.0 1C (red), 28.5 1C (orange), 31.5 1C (green), 34.0 1C (blue), 38.0 1C(purple) and 39.5 1C (brown). (C) Characteristic length scales of the self-assembled PDMS droplets from Lc = 2p/qm, where qm is obtained from peaks inI(q). Error bars in (B and C) are standard deviations from 3 independent measurements within a sample. (D) Zoomed out image in the xz-plane of ahydrogel scaffold printed at T = 34.0 1C. Dashed line indicates boundary between two printed layers. Inset: Zoomed in image of the same sample.


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length scale, Lc = 2p/qm, is defined by the local peak qm in the I(q)plots where possible and is plotted in Fig. 6C. The value of Lc

increases gradually to a maximum at T = 34.0 1C, followed by adecrease that is brought on by kinetic arrest at highertemperatures.16 These results are in agreement with our earlierwork on a similar system of thermoresponsive nanoemulsions inwhich samples were cast into molds.39 In addition, we verify thatthe connective mesoscale structure is preserved between eachprinted layer. A zoomed out CLSM image of the hydrogel scaffoldin the direction perpendicular to the printing (xz-plane) is shownin Fig. 6D, where a dashed line indicates the boundary of the twolayers as observed in HRSEM images in Fig. 8A.

The interconnectivity of the gel networks formed by the self-assembled thermoresponsive nanoemulsions allows for thetransport of molecules and mesoscopic objects. We demonstratethis capability by using the nanoemulsions as a sacrificialtemplate that is extracted from the 3D printed hydrogels throughsolvent transfer with pure toluene. After rehydration, we immersethe samples in aqueous solutions containing fluorescent,carboxylated polystyrene beads with diameters of 50 nm, 200 nm,and 1.0 mm. Fig. 7 illustrates the capability of the printed poroushydrogels to serve as size-selective membranes. Beads of allsizes are able to diffuse into hydrogels having comparativelylarger pore size (T = 34.0 1C and 38.0 1C) but not those withsmaller pores (T = 28.5 1C, 31.5 1C, 39.5 1C). The control scaffoldsample printed at T = 22.0 1C (T o Tgel) does not permit thepassage of the smallest bead size (50 nm) because of the isolatedpore structure within it. A representative 3D CLSM image of aporous sample printed at T = 34.0 1C and treated with 200 nmbeads shows that the fluorescent beads are able to diffuse intothe channels in x, y, and z-directions (Fig. 8B). The xz-imageof a porous sample (T = 34.0 1C) in Fig. 8C is captured from anxyz-stack; here, the imaging height is limited to 24 mm due tosignificant scattering from the remainder of the sample.

Fig. 7 Printed porous hydrogels as size-selective membranes. Representative CLSM images of mesoporous hydrogel samples in which fluorescentpolystyrene beads have been introduced. Beads of diameters (A) 50 nm, (B) 200 nm and (C) 1.0 mm are allowed to diffuse into the interconnected porousnetwork. Fluorescence here indicates the passage of beads into the sample. All images are captured at z = 10 mm above the bottom of the sample.

Fig. 8 Images of printed hydrogels in three dimensions. (A) HRSEM ofporous hydrogel scaffold (T = 34.0 1C) in the xz-plane, showing layeredstructure from SLA printing. Arrows indicate approximate location ofboundaries spaced 100 mm apart. (B and C) CLSM images of the samescaffold treated with fluorescent polystyrene beads (2a = 200 nm) in the(B) xy-plane and (C) xz-plane. The images in (B and C) are captured asan xyz-stack, where the z-dimension is limited to 24 mm because ofsignificant backscatter from the rest of the sample.

Fig. 9 Mechanical properties of filled and porous hydrogel scaffolds.(A) Young’s modulus and (B) engineering rupture stress of the filled (solid)and porous (open) as a function of T. Error bars are standard deviationsfrom 6 independent samples.


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An additional observation is that the Young’s modulus andengineering rupture stress for hydrogels with and withoutnanoemulsions remain similar to within the experimentaluncertainty (Fig. 9). This suggests that the mechanical propertiesof the hydrogel can be tuned completely independently from theinternal architecture necessary for diffusion of nutrients or cellpassage, which could be important in future applications of thismaterial towards synthetic microvasculature and 3D cellularscaffolds.

4. Conclusions

Thermoresponsive nanoemulsions belong to a class of stimuli-responsive materials that are compatible with 3D SLA printingto generate hierarchical materials with tunable mesoscalestructures. The PEGDMA molecules in the nanoemulsion inkserve two important functions: (1) they induce colloidal gelationin which the characteristic length scale can be tuned based onthe temperature, and (2) they provide photochemically cross-linked hydrogel materials on demand. Using a commerciallyavailable 3D printer with modifications to the resin tray toenhance oxygen permeability, we demonstrate that these nano-emulsions can serve as self-assembling precursor inks to berapidly photopolymerized into macroscale shapes with thedesired internal features. Correct selection of the photoinitiatoris critical to maintain the stability of the nanoemulsions overthe course of the printing process and to provide photoreactiveresponse with the near-UV (l = 405 nm) light sources that arecommonly used in commercial 3D SLA printers. In addition, therapid structural recovery and shear thinning rheological propertiesof the ink is necessary for generating hierarchical materials withan interconnected morphology that allows the mass transport ofmesoscopic objects into the hydrogels. However, due to the yieldstress behavior of these inks, wiper operations between successiveprint steps are needed to spread the ink out evenly.

Future possibilities for these mesoporous materials includethe crystallization of hydrophobic molecules in nanoemulsion-carrying channels for controlled or triggered drug delivery,40

synthetic vasculature,41 or in understanding the deformationmechanics of tissue-like models that can be printed in multipleshapes and sizes.42 Other applications involve retaining thenanoemulsions in the printed structure to serve as reservoirs\for hydrophobic drugs,43 modulating the mass transfer of gases,44

or acting as sinks for metals/organics in water treatment processes.45

Competing financial interest

The authors declare no competing financial interest.

Acknowledgements

This work was supported primarily by the MRSEC Program ofthe National Science Foundation under DMR – 1419807. Theauthors thank Prof. G. McKinley for the use of the rheometerand J. F. Hamel for loan of the environmental incubator.

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